Examples of a real time polymerase chain reaction (real time PCR) method that is widely used to detect a gene include a TaqMan (Registered Trademark) method and an SYBR (Registered Trademark) Green method.
The TaqMan (Registered Trademark) method, which is an extremely highly sensitive method, has (i) a problem of complexity of designing and synthesis of a probe for use in detection and (ii) a problem of high detection cost.
Meanwhile, the SYBR (Registered Trademark) Green method is a convenient method using a pigment that increases in fluorescence intensity when bound to a double-stranded DNA. Note, however, that according to the SYBR (Registered Trademark) Green method, a nonspecifically amplified double-stranded DNA will also be detected as “positive. In view of this, the SYBR (Registered Trademark) Green method has a problem of causing a great error in detection. Further, the SYBR (Registered Trademark) Green method also has a problem of difficulty in designing of a primer having high detection accuracy.
In order that the problems of the TaqMan (Registered Trademark) method and of the SYBR (Registered Trademark) Green method will be solved, new PCR methods have been developed so far (see, for example, Patent Literature 1).
The following description discusses, with reference to FIG. 26, a basic concept of a technique disclosed in Patent Literature 1.
The technique disclosed in Patent Literature 1 uses a primer that is capable of forming a double strand in a molecule and is also capable of forming a cytosine bulge structure in the double strand, the primer emitting fluorescence in response to binding of 2,7-diamino-1,8-naphthyridine (DANP) to the cytosine bulge structure. Note that a cytosine bulge structure is a characteristic structure formed by cytosine with which no complementary nucleotide is to be paired.
Before a PCR reaction is started, DANP binds to the cytosine bulge structure in the primer, and intense fluorescence is emitted (see (d) of FIG. 26).
After the PCR reaction is started, first, the primer binds to a template, and a complementary strand of the template is extended from the primer (see (a) of FIG. 26).
Next, a double strand formed by the complementary strand and the template is separated, by a high temperature treatment, into (a) a single strand including the complementary strand (hereinafter referred to as a “single strand A”) and (b) a single strand including the template.
Subsequently, another primer (not illustrated in FIG. 26) binds to the single strand A, and the complementary strand of the single strand A is extended from the another primer (see (b) of FIG. 26).
During a process in which the complementary strand of the single strand A is extended, a complementary nucleotide corresponding to the cytosine is formed in the complementary strand of the single strand A. This results in a loss of the cytosine bulge structure (see (c) of FIG. 26).
According to the technique disclosed in Patent Literature 1, the number of cytosine bulge structures decreases as the PCR reaction progresses, so that a fluorescence intensity is reduced (see (d) of FIG. 26). According to the technique disclosed in Patent Literature 1, the PCR reaction is detected by observing a reduction in fluorescence intensity.